【摘要】 目的:构建Ki-ras-PET-28α(+)原核表达质粒,并表达、纯化得到Ki-P21ras蛋白。方法:人工合成Ki-ras基因cDNA的蛋白编码CDs区序列,并在其正义链5’加上BamHⅠ酶切位点,3’加上HindⅢ酶切位点,经过BamHⅠ和HindⅢ双酶切后纯化回收得到具有粘性末端的Ki-rascDNA,将PET-28α(+)同样经过BamHⅠ和HindⅢ双酶切后纯化回收得到与Ki-rascDNA有相同粘性末端的线形片段,通过T4DNA连接酶将具有粘性末端的Ki-rascDNA与酶切后的PET-28α(+)定向连接,构建重组质粒Ki-ras-PET-28α(+),经酶切和测序鉴定。将鉴定正确的Ki-ras-PET-28α(+)转入感受态细菌BL21(DE3)中诱导表达,利用组氨酸"标签"(His-Tag)对P21ras进行亲和层析纯化。结果:测序分析证实,克隆人PET-28α(+)的Ki-ras序列与Genbank登录号为"M54968"的Ki-rascDNA序列一致,将重组质粒Ki-ras-PET-28α(+)转化BL21(DE3),IPTG诱导目的蛋白表达。SDS-PAGE和蛋白纯化结果表明,Ki-ras-PET-28α(+)在大肠杆菌中获得可溶性高表达,经Ni2+-NTA-树脂柱亲和层析纯化得到了PAGE纯的ki-P21ras蛋白,Western-Blot分析证实其免疫活性。结论:成功构建了原核表达载体Ki-ras-PET-28α(+),并经表达、纯化得到具有生物活性的P21ras蛋白,为研究P21ras在肿瘤发病中的作用和机制以及抗P21ras细胞内抗体的研究奠定了基础。更多还原

【Abstract】 Objective:To construct the prokaryotic expression plasmid Ki-ras-PET-28α(+) and to obtain the purified protein of P21ras. Methods:After the CDs of Ki-ras cDNA with enzyme site BamHⅠand HindⅢ were artificially synthesized,Ki-ras was digested by BamHⅠand HindⅢ ,and directly ligated into BamHⅠ/HindⅢ-digested PET-28α(+).The recombinant plasmid was identified by gel electrophoresis and sequencing and named as Ki-ras-PET-28α(+).The recombinant plasmid was transformed into BL21(DE3),which was induced by IPTG. With an His-tag sequence,the recombinant protein possessed the affinity to Ni2+,thus the target protein could be purified by Ni2+-NTA-resin column.The protein was analyzed by SDS-PAGE and sequence analysis.Results:Sequence analysis showed that the Ki-ras sequence of Ki-ras-PET-28α(+) was identical to that in GeneBank (accession M54968).Also,the expression and purification of P21ras were confirmed successfully by SDS-PAGE and Western-blotting.Conclusion:The prokaryotic expression plasmid(Ki-ras-PET-28α(+))has been successfully constructed, expressed and purified ,which provides a basis for further researching the nosogenesis of carcinoma and developing the antibody of P21ras.更多还原